2 2 azinobis 3 ethylbenzothiazoline sulfonic acid abts

Tart cherries were purchased at a local market, pits were removed, and flesh was used for the preparation of jams. The following chemicals were used: Potassium chloride, sodium acetate, hydrochloric acid, methanol, sodium carbonate, sodium bisulfite, Folin-Ciocalteu reagent were purchased from Kemika (Zagreb, Croatia). Trolox, 2,2’-azinobis (3-ethylbenzothiazoline sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The immunoenzymatic assays were carried out to detect antibody reactivity against STAg or recombinant proteins in serum samples from chickens, pigs, mice, and humans. Briefly, high affinity polystyrene microtiter plates (Corning 3590 Laboratories, Inc., New York, NY, USA) were coated with STAg (10 mg/ml) or recombinant proteins (2 mg/ml) in carbonate-bicarbonate buffer 0.06 M (pH 9.6) for 18 h at 4°C. Plates were incubated with serum samples and subsequently with secondary antibody specific to each species conjugated to peroxidase (Sigma). The assay was revealed by adding the enzyme substrate (0.03% H₂O₂ and 2,2-azino-bis-3-ethyl-benzothiazoline sulfonic acid [ABTS, Sigma] in 0.07 M citrate phosphate buffer, pH 4.2). The cut off of the reaction was calculated as the mean OD of negative control sera plus 3 standard deviations. The negative control sera consisted of a pool of 10 serum samples tested negative for T. gondii infection. Antibody titers were expressed as ELISA index (EI), according to the following formula: EI = OD sample/OD cut off, as previously described (Silva et al., 2002 (link)). Samples with EI values ≥ 1.2 were considered positive.

In this study, the following reagents and standards were used: aluminium chloride, quercetin dihydrate, gallic acid monohydrate, potassium persulfate, Trolox, 2,2-azinobis(3-ethylbenzothiazoline sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazil (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ) were obtained from Sigma-Aldrich (St. Lois, MO, USA); HPLC standards (gallic and caffeic acid, (+)-catechin hydrate, (−)-epicatechin, rutin hydrate, quercetin) were purchased from Sigma-Aldrich Chemie Gmbh (Steinheim, Germany), and malvidin 3-glucoside from Extrasynthese (Genay, France). Folin–Ciocalteu reagent, sodium nitrite, sodium carbonate, sodium hydroxide, potassium bisulphite, sodium acetate, potassium chloride and hydrochloric acid were purchased from Kemika (Zagreb, Croatia); sodium acetate trihydrate, ferric chloride hexahydrate, and ammonium acetate were purchased from Gram-Mol (Zagreb, Croatia); copper(II) chloride from Acros Organics (New Jersey, NJ, USA); HPLC grade methanol and neocuproine from Merck (Darmstadt, Germany) and phosphoric acid (HPLC grade) were obtained from Fluka (Buchs, Switzerland).